umkhiqizo

1. Isendlalelo

I-Tylosiniyi-macrolide antibiotic, esetshenziswa kakhulu njenge-antibacterial kanye ne-anti-mycoplasma.Ama-MRL aqinile awakasungulwa njengoba lesi sidakamizwa singase siholele kumphumela omubi emaqenjini athile.

Le khithi ingumkhiqizo omusha osuselwe kubuchwepheshe be-ELISA, obusheshayo, obulula, obunembile futhi obuzwelayo uma uqhathaniswa nokuhlaziywa kwezinsimbi okuvamile futhi udinga amahora angu-1.5 kuphela ekusebenzeni okukodwa, kunganciphisa kakhulu iphutha lokusebenza kanye nokuqina komsebenzi.

2. Isimiso Sokuhlola

Le khithi isuselwe kubuchwepheshe obungaqondile bokuncintisana be-ELISA.Imithombo ye-microtiter ihlanganiswe ne-antigen ehlangene.Izinsalela ze-Tylosin kusampula ziqhudelana ne-antigen eboshwe epuleti le-microtiter le-antibody.Ngemva kokwengezwa kwe-enzyme ebizwa ngokuthi i-anti-antibody, i-TMB substrate isetshenziselwa ukukhombisa umbala.Ukungabi khona kwesampula kuhlobene kabi ne-tylosin ehlala kuyo, ngemva kokuqhathanisa ne-Standard Curve, ephindaphindwa nge-dilution factor, inani lensalela ye-tylosin kusampula lingabalwa.

3. Izicelo

Le kit ingasetshenziswa ekuhlaziyweni komthamo kanye nekhwalithi yezinsalela ze-tylosin ezicutshini zezilwane (inkukhu, ingulube, idada) nobisi, uju, iqanda, njll.

4. Ukusabela okuphambene

I-Tylosin……………………………………………………..100%

I-Tilmicosin…………………………………………………<2%

5. Izinto Ezidingekayo

5.1 Izisetshenziswa:

---- I-Microtiter plate spectrophotometer (450nm/630nm)

----I-Rotary evaporator noma amathuluzi okomisa i-nitrogen

-------homogenizer

-----Shaker

---- I-Centrifuge

----Ibhalansi yokuhlaziya (i-inductance: 0.01g)

---- I-pipette ethweswe iziqu: 10ml

---- I-rubber pipette bulb

-----I-Volumetric flask: 10ml

----Amashubhu e-Polystyrene centrifuge: 50ml

Ama-Micropipettes: 20-200ml, 100-1000ml

250ml-multipipette

5.2 Ama-reagents:

----I-sodium hydroxide (NaOH, AR)

-----Sodium bicarbonate (NaHCO_3,AR)

---- I-Sodium carbonate (NaCO₃, AR)

---- I-Trichloroacetic acid (AR)

---- I-Acetonitrile (AR)

----I-Ethyl acetate (AR)

┅┅n-Hexane (AR)

---- Amanzi akhishiwe

6. Izingxenye zekhithi

l Ipuleti leMicrotiter elinemithombo engama-96 embozwe nge-antigen

l Izixazululo ezijwayelekile (amabhodlela ama-5, 1ml/ibhodlela)

0ppb, 0.5ppb, 1.5ppb, 4.5ppb, 13.5ppb

l Ukulawula okujwayelekile kwe-Spiking: (1ml/ibhodlela)1 ppm

l I-enzyme conjugate 1ml……………..…..…ikepisi elibomvu

l Isixazululo se-antibody 7ml……………….……ikepisi eliluhlaza

l Isixazululo A 7ml…………………….….……ikepisi elimhlophe

l Isixazululo B 7ml………………………………..ikepisi elibomvu

l Isixazululo sokumisa 7ml.…………….……….ikepisi eliphuzi

l 20 × isixazululo esigxilile sokugeza 40ml

……………………………………………ikhephu esobala

l 4 × isixazululo kanzulu isizinda 50ml

………………………………………………….ikepisi eliluhlaza okwesibhakabhaka

7. Ukulungiselela ama-reagents:

Isixazululo 1:Isixazululo se-NaOH esingu-0.1mol/L

Kala u-0.4g NaOH ukuya ku-100ml wamanzi akhishiwe bese uxuba ngokuphelele.

Isixazululo 2: 1mol/L isixazululo se-NaOH

Kala u-4g NaOH ukuya ku-100ml wamanzi akhishiwe bese uwaxuba ngokuphelele.

Isixazululo 3: Usawoti we-carbonate buffer salt

Isixazululo1: 0.2M PB

Chaza u-51.6g we-Na₂I-HPO₄·12H₂O, 8.7g ye-NaH₂PO₄·2H₂O ngamanzi e-deionized bese unciphisa ku-1000ml.

Isixazululo2: Isixazululo sokukhipha

Nciphisa isixazululo se-2× esigxilisiwe sokukhipha ngamanzi a-deionized ngesilinganiso sevolumu 1:1(isib. 10ml ka-2× wesixazululo sokukhipha + 10ml wamanzi akhishiwe), ezosetshenziselwa ukukhipha isampula,lesi sixazululo singagcinwa ku-4 ℃ inyanga engu-1.

Isixazululo3: Isixazululo sokugeza

Nciphisa isixazululo sokugeza esingu-20× esigxilisiwe ngamanzi akhishwe nge-deionized ngesilinganiso sevolumu engu-1:19(isib. 5ml ka-20×isixazululo sokugeza + 95ml wamanzi akhishiwe), ezosetshenziselwa ukugeza amapuleti.Lesi sixazululo singagcinwa ku-4 ℃ inyanga engu-1.

8. Isampula Amalungiselelo

8.1 Isaziso kanye nezinyathelo zokuqapha ngaphambi kokusebenza:

(a) Sicela usebenzise amathiphu aphuma kanye kunqubo yokuhlola, futhi ushintshe amathiphu lapho uthatha i-reagent ehlukile.

(b) Qiniseka ukuthi zonke izinsimbi zihlanzekile.

(c) Gcina amasampula ezicubu emakhazeni.

(d) Isampula elungisiwe kufanele isetshenziselwe ukuhlola ngesikhathi esisodwa.

8.2 Izicubu zezilwane (inkukhu, ingulube, njll)

-----Homogenize isampula nge-homogenizer;

----Thatha u-2.0±0.05g we-homogenate uye ku-50ml polystyrene centrifuge tube;engeza u-2ml ka-0.2M PB (isisombululo1) , qhaqhazela ukuze uncibilike, bese wengeza u-8ml we-ethyl acetate bese unyakazisa kakhulu 3min;

---- I-Centrifuge yokuhlukanisa: 3000g / izinga lokushisa elizungezile / 5min.

----Dlulisela u-4ml wesigaba sezinto eziphilayo ezingaphezu kuka-10ml, omiswe ngamanzi okugeza angu-50-60℃ ngaphansi kwegesi ye-nitrogen;

----Chaza insalela eyomile ngo-1ml we-n-hexane, i-vortex for 30s ukuze incibilike, bese wengeza u-1ml wesixazululo sokukhipha (isisombululo2), i-vortex ye-1min.i-centrifuge yokuhlukanisa: 3000g / izinga lokushisa elizungezile / 5min

---- Susa isigaba se-n-hexane esinamandla amakhulu;thatha u-50μl wesigaba se-aqueous substrate ukuze uhlole.

I-Dilution factor: 1

8.2 Ubisi

----Thatha u-100μl wesampula yobisi olungaphekiwe, xuba no-900μl wesixazululo sokukhipha (isisombululo2), bese uxuba ngokuphelele.

----Thatha u-50μl wesixazululo esilungisiwe sokuhlola.

I-Dilution factor: 10

9. Inqubo yokuhlola

9.1 Qaphela ngaphambi kokuhlolwa

9.1.1Qinisekisa ukuthi wonke ama-reagents nama-microwells konke kusezingeni lokushisa elilingana negumbi (20-25℃).

9.1.2Buyisela wonke ama-reagents asele ku-2-8℃ngokushesha ngemva kokusetshenziswa.

9.1.3Ukugeza ama-microwells ngendlela efanele kuyisinyathelo esibalulekile enqubweni yokuhlola;kuyisici esibalulekile ekukhiqizeni kabusha kokuhlaziywa kwe-ELISA.

9.1.4 Avala ukukhanya futhi umboze ama-microwell ngesikhathi sokufukamela.

9.2 Izinyathelo Zokuhlola

9.2.1 Khipha wonke ama-reagents ekamelweni lokushisa (20-25℃) ngaphezu kwemizuzu engama-30, qhaqhazela kancane ngaphambi kokuwasebenzisa.

9.2.2 Khipha ama-microwell adingekayo bese ubuyisela amanye esikhwameni se-zip-lock ku-2-8℃ ngokushesha.

9.2.3 Ingxube yokugeza ehlanjululwe kufanele ifudunyezwe kabusha ukuze ibe sezingeni lokushisa elilingana negumbi ngaphambi kokusetshenziswa.

9.2.4Inombolo:Ifakwe izinombolo kuzo zonke izindawo ze-microwell nawo wonke amazinga namasampuli kufanele asetshenziswe ngokuphindwe kabili.Rekhoda amazinga kanye nezikhundla zamasampuli.

9.2.5Add isixazululo esijwayelekile/isampula nesisombululo se-antibody: Engeza u-50µl wesixazululo esijwayelekile((ikhithi enikeziwe)) noma isampula elilungiselelwe emithonjeni ehambisanayo.Engeza u-50µl wesisombululo se-antibody(ikhithi enikeziwe).Hlanganisa kahle ngokunyakazisa ipuleti ngesandla bese ufukamela imizuzu engama-30 ku-37℃ ngekhava.

9.2.6Geza: Khipha ikhava ngobumnene futhi uhlanze uketshezi oluphuma emithonjeni bese uhlanza ama-microwells ngesisombululo sokugeza esihlanjululwe esingu-250µl (isisombululo3) ngezikhathi ezingu-10 izikhathi ezingu-4-5.Gcoba amanzi asele ngephepha elimuncayo (ibhamuza lomoya elisele lingaqedwa ngethiphu elingasetshenzisiwe).

9.2.7Engeza i-enzyme conjugate: Engeza i-100ml yesisombululo se-enzyme conjugate (ikhithi enikeziwe) emthonjeni ngamunye, xuba ngobumnene futhi ufukamele imizuzu engama-30 ku-37℃ ngekhava.Phinda isinyathelo sokugeza futhi.

9.2.8Umbala: Engeza u-50µl wesixazululo A(ikhithi enikeziwe) kanye no-50µl wesixazululo B(ikhithi enikeziwe) emthonjeni ngamunye.Hlanganisa ngobumnene bese ufukamela imizuzu eyi-15 ku-37℃ ngekhava.

9.2.9Kala: Engeza u-50µl wesixazululo sokumisa(ikhithi enikeziwe) emthonjeni ngamunye.Xuba ngobumnene futhi ulinganise ukumunca ku-450nm (Kuyaphakanyiswa isilinganiso esinobude obumbili begagasi obungu-450/630nm. Funda umphumela phakathi kwemizuzu emi-5 ngemva kokwengeza isixazululo sokumisa).

10. Imiphumela

10.1 Iphesenti lokumunca

Amanani amaphakathi wamanani okumunca atholwe kumazinga kanye namasampuli ahlukaniswa ivelu yokumunca yezinga lokuqala (izinga elinguziro ) futhi liphindwe ngo-100%.Izinga elinguziro lenziwa lalingana no-100% futhi amanani okumunca acashunwe ngamaphesenti.

B

Ukungabi nalutho (%) = —— × 100%

B0

B ——izinga le-absorbance (noma isampula)

B0 ——absorbance zero standard

10.2 Ijika Elijwayelekile

----Ukuze udwebe ijika elijwayelekile: Thatha inani lokumunca lamazinga njenge-y-eksisi, i-semi logarithmic yokugxiliswa kwesixazululo sezindinganiso ze-tylosin (ppb) njenge-x-eksisi.

----Ukugxiliswa kwe-tylosin kwesampula ngayinye (ppb), engafundwa kwijika lokulinganisa, kuphindwa nge-Dilution factor ehambisanayo yesampula ngayinye elandelwayo, futhi ukugxiliswa kwangempela kwesampula kuyatholakala.

Sicela uqaphele:

isofthiwe ekhethekile yenzelwe ukuhlaziywa kwedatha, enganikezwa ngokucela.

11. Ukuzwela, ukunemba nokunemba

Ukuzwela Kokuhlola:1.5ppb

Umkhawulo wokutholwa:

Izicubu zezilwane………………………………………………1.5ppb Ubisi…………………………………………………………..15ppb Ukunemba:

Izicubu zezilwane……………………………………………80±15%

Ubisi…………………………………………………..80±10%

Ukunemba:

I-Coefficient ehlukile yekhithi ye-ELISA ingaphansi kuka-10%.

12. Qaphela

12.1 Amanani amaphakathi wamanani okumunca atholwe kumazinga kanye namasampula azoncishiswa uma ama-reagents namasampuli engalawulwanga kumazinga okushisa egumbi (20-25℃).

12.2 Ungavumeli ama-microwells ome phakathi kwezinyathelo ukugwema ukukhiqizwa kabusha okungaphumeleli futhi usebenzise isinyathelo esilandelayo ngokushesha ngemva kokuthepha isibambi sama-microwells.

12.3 Nyakazisa i-reagent ngayinye ngobumnene ngaphambi kokuyisebenzisa.

12.4 Gcina isikhumba sakho kude nesixazululo sokumisa ngoba singu-0.5MH₂SO₄isisombululo.

12.5 Ungawasebenzisi amakhithi asephelelwe yisikhathi.Ungashintshisani ama-reagents amaqoqo ahlukene, kungenjalo kuzokwehlisa ukuzwela.

12.6 Gcina amakhithi e-ELISA eku-2-8℃, ungafrizi.Vala amapuleti amancane, Gwema ukukhanya kwelanga okuqondile phakathi nawo wonke ama-incubation.Kunconywa ukumboza amapuleti e-microtiter.

12.7 Isixazululo se-substrate kufanele siyekwe uma sishintsha imibala.Ama-reagents angase abe mabi uma inani lokumunca (450/630nm) lezinga elinguziro lingaphansi kuka-0.5 (A450nm<0.5).

12.8 Ukusabela kombala kudinga imizuzu engu-15 ngemva kokwengezwa kwesixazululo A kanye nekhambi B. Futhi ungakwazi ukwelula ububanzi besikhathi sokufukamela ukuya kumaminithi angu-20 noma ngaphezulu uma umbala ulula kakhulu ukuthi unganqunywa.Ungalokothi weqe imizuzu engama-30, kunalokho, finyeza isikhathi sokufukamela ngendlela efanele.

12.9 Izinga lokushisa elilungile lokusabela ngu-37℃.Izinga lokushisa eliphakeme noma eliphansi lizoholela ekushintsheni kokuzwela kanye namanani okumunca.

13. Isitoreji

Isimo sesitoreji: 2-8℃.

Isikhathi sokugcina: izinyanga eziyi-12.